Purification of E. coli invasin IbeA-binding protein in intestinal epithelial cells.
- Author:
Chang-ye HUI
1
;
Yan GUO
;
Jun LI
;
Xiao-yan HAO
;
Hong CAO
;
Sheng-he HUANG
Author Information
- Publication Type:Journal Article
- MeSH: Caco-2 Cells; Epithelial Cells; cytology; metabolism; microbiology; Escherichia coli Proteins; genetics; isolation & purification; metabolism; Humans; Intestines; cytology; metabolism; Membrane Proteins; genetics; isolation & purification; metabolism; Recombinant Proteins; genetics; isolation & purification
- From: Journal of Southern Medical University 2009;29(12):2375-2378
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo purify IbeA-binding protein from intestinal epithelial Caco-2 cells.
METHODSRecombinant IbeA was purified, and 1, 5, and 10 microg/ml His-IbeA and bovine serum albumin (control) were preincubated with confluent Caco-2 monolayer for 30 min at 4degrees celsius;. Gentamicin protection assay was used to test the invasion of E. coli K1 pathogenic isolate E44 in Caco-2 cells. The binding proteins were purified from Caco-2 by IbeA-Cu(2+) sepharose affinity chromatography, and validated by Far-Western blotting. The N-terminal amino acid sequence of the binding protein was determined using Edman assay.
RESULTSE44 invasion in Caco-2 cells was blocked by the recombinant IbeA in a dose-dependent manner. Two binding bands were obtained with His pull-down, and the binding specificity was demonstrated by Far-Western blotting. The N-terminal amino acid sequence of IBP200 was MASITKLP with an isoelectric point of about 5.0.
CONCLUSIONTwo novel Caco-2 proteins interacting with IbeA of E. coli have been purified and identified.